来源于Escherichia coli的高比活植酸酶基因的高效表达  

Overexpression of Escherchia coli Phytase with High Specific Activity

作　　者：罗会颖[1] 姚斌[1] 袁铁铮[1] 王亚茹[1] 史秀云[1] 伍宁丰[2] 范云六[2] 

机构地区：[1]中国农业科学院饲料研究所,北京100081 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《生物工程学报》2004年第1期78-84,共7页Chinese Journal of Biotechnology

基　　金：国家高技术研究与发展计划 ( 863计划 )项目资助 (No .2 0 0 1AA2 14 0 41)~~

摘　　要：高效表达高比活植酸酶是进一步提高植酸酶发酵效价、降低植酸酶生产成本的一个有效途径。对源于Escherichiacoli的高比活植酸酶基因appA ,按照毕赤酵母 (Pichiapastoris)密码子的偏爱进行了密码子优化改造。该改造后的基因appA m按正确的阅读框架融合到毕赤酵母表达载体pPIC9上的α 因子信号肽编码序列 3′端 ,通过电击转化得到重组转化子。对重组毕赤酵母的Southernblotting分析证实植酸酶基因已整合到酵母基因组中 ,并确定了整合基因的拷贝数。Northernblotting分析证实植酸酶基因得到了正常转录。SDS PAGE分析和表达产物的研究表明 ,植酸酶得到了高效分泌表达 ,在 5L发酵罐中植酸酶蛋白表达量达到 2 5mg mL发酵液 ,酶活性 (发酵效价 )达到 7 5× 10 6 IU mL发酵液以上 ,大大高于目前报道的各种植酸酶基因工程菌株的发酵效价。High-level expression of phytase with high specific activity is an effective way to improve phytase fermentation potency and reduce its production cost. The gene appA encoding Escherchia coli phytase AppA with high specific activity was modified and artificially synthesized according to the bias in codon choice of the high expression gene in Pichia pastoris without changing the amino acid sequence of the AppA. The modified gene, appA-m, was inserted in the Pichia pastoris expression vector pPIC9, then introduced into the host Pichia pastoris by electroporation. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. The result of Southern blotting analysis of the recombinant yeast indicated that only one copy of the appA-m gene was integrated into the genome of Pichia pastoris. The result of Northern analysis of the recombinant yeast showed that the modified gene was effectively transcribed. SDS-PAGE analysis of the phytase expressed in Pichia pastoris revealed that the phytase was overexpressed and secreted into the medium supernatant. There are three phytase proteins with apparent molecular weight in approximately 50kD, 52kD and 54kD respectively in the media, which are larger in the size than the native phytase from E.coli. The results of N-terminal sequecing and deglycosylation of the expressed phytase in Pichia pastoris proved that the expressed phytase were glycosylated protein with different glycosylation degree. The expressed phytase Pichia pastoris shared similar pH and temperature optima to those of the natural phytase from E.coli and had highly resistant to pepsin digestion. In 5-L fermentor, after induced by 0.5% methanol for 120 h, the expression level of phytase protein was 2.5mg/mL, and the phytase activity (fermentation potency) exceeded 7.5×106 IU/mL, which was the highest among those of all kinds of recombinant strains reported now.

关 键 词：高比活植酸酶 基因改造 毕赤酵母 基因表达 

分 类 号：Q786[生物学—分子生物学] Q814